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Positive Direct Coombs Test Induced by Phenylhydrazine

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Positive Direct Coombs Test Induced by Phenylhydrazine Positive Direct Coombs Test Induced by Phenylhydrazine E. E. Muirhead, … , M. Groves, S. Bryan J Clin Invest. 1954;33(12):1700-1711. https://doi.org/10.1172/JCI103050. Research Article Find the latest version: https://jci.me/103050/pdf POSITIVE DIRECT COOMBS TEST INDUCED BY PHENYLHYDRAZINE 1 By E. E. MUIRHEAD, M. GROVES, AND S. BRYAN (Froim the Department of Pathology, Southwestern Medical School of the University of Texas. Dallas, Tex.) (Submitted for publication April 9, 1954; accepted July 21, 1954) The Coombs or antiglobulin test was introduced in 95 per cent ethyl alcohol and the percentage concen- for the detection of antibodies to erythrocytes of tration of erythrocytes containing these structures was a type termed "incomplete antibodies" (1). The obtained by counting 1000 erythrocytes (27). The me- chanical fragility of erythrocytes was determined by a indirect Coombs test has gained wide application modification of the method of Shen, Castle, and Fleming in the detection of the antibodies to various eryth- (28). One cc. of oxalated blood (5 cc. blood plus 10 rocytic antigens (2) and in the cross-matching of mg. of a buffered mixture of potassium oxalate and am- blood for transfusions (3). The direct Coombs monium oxalate) was placed in a 25 cc. Erlenmeyer flask test has become a diagnostic and investigative tool containing 20 glass beads, each of about 4 mm. diameter. The flask was clamped to a plastic disc 16.5 cm. from in hemolytic disease of the newborn (4-6), fol- the axis and rotated for 90 minutes at 30 rpm. at room lowing incompatible blood transfusions (7) and temperature. The amount of hemoglobin in the superna- in the acquired hemolytic anemias, idiopathic or tant plasma was related to the total hemoglobin concen- secondary types (8-22). tration of the blood as per cent hemolysis. All of these conditions in man when associated The plasma hemoglobin concentration was determined by the method of Bing and Baker (29). The serum bili- with positive direct and indirect Coombs tests, rubin concentration was determined by the method of have been interpreted to be the result of immune Malloy and Evelyn (30). mechanisms, either of the isoimmune or autoim- The Coombs tests: The canine serum anti-serum was mune type. In the present communication the pro- prepared in the rabbit by slight modifications of the di- duction of a positive direct Coombs test in the rections of Mourant (31): Rabbits weighing 2 to 3 kg. were injected with pooled canine serum obtained by mix- dog by means of the drug phenylhydrazine is de- ing about 5 cc. of serum from each of three to five dogs. scribed. The phenomenon is unattended by the A course of intravenous injections was given at 2-to-3- immunologic implications of blood incompatibility day intervals beginning with a 0.5 cc. dose and following and is not associated with the usual conditions with 5 doses of 1 cc. each. The animal was bled 10 a or days after the last dose. Pooled erythrocytes from nor- causing secondary symptomatic acquired he- mal dogs were used in absorbing the rabbit serum. The molytic anemia. serum was then passed through a Seitz filter and ali- quots of 1 cc. were stored in small sterile bottles at MATERIAL AND METHODS -20'C. until used. The results with this serum were on nine occasions with the results a Adult normal mongrel dogs of either sex weighing compared using canine serum furnished Drs. L. E. 5.5 to 11 kg. were used. After the control observations antiglobulin kindly by had been completed, a 1 to 2 per cent solution of phenyl- Young and S. N. Swisher of Rochester, New York. hydrazine hydrochloride (Merck) in saline was injected When these two sera were used undiluted with cells giv- intravenously in a single dose of 40 mg. per kg. body ing positive results, the degree of agglutination evoked by weight. The studies were conducted periodically there- the two was identical. after up to 41 days. In the direct Coombs test one drop of a 2 per cent The hemoglobin concentration of the peripheral blood suspension of the canine erythrocytes was washed three was determined by the alkaline hematin method (23). times in abundant saline at room temperature. After the The hematocrit was determined according to Wintrobe third wash the test tube was turned up and allowed to (24) and the reading was corrected for trapped plasma drain. To the erythrocytes suspended in the residual according to the method of Chaplin and Mollison (25). saline, a suspension approximating the original 2 per cent Reticulocytes were estimated routinely by counting 1000 concentration, 2 drops of the canine serum anti-serum erythrocytes stained with brilliant cresyl blue (26). (Coombs serum) were added. The mixture was al- Heinz bodies were stained with 0.2 per cent methyl violet lowed to stand for 30 minutes following which it was centrifuged for one minute at 500 to 1000 rpm. After 1 Supported by a grant from the U. S. Public Health shaking the button of cells into the supernatant the re- Service, National Heart Institute. sults were evaluated grossly as follows: a solid button 1700 POSITIVE DIRECT COOMBS TEST INDUCED BY PHENYLHYDRAZINE 1701 of agglutinated cells was designated 4 +, a few large The indirect Coombs test was conducted by incubating clumps 3 +, multiple scattered smaller clumps 2 +, a erythrocytes from normal dogs as a 2 per cent suspen- definitely granular suspension with fine clumps 1+. All sion at 370 C. for 30 minutes in the serum of the dogs of these grossly detectable degrees of agglutination were receiving phenylhydrazine and then treating the cells as checked by microscopic inspection which also demon- in the direct procedure. A random panel of canine strated that the clumps of erythrocytes were stable for erythrocytes obtained from three normal dogs was used periods longer than 5 minutes. A smooth or fairly throughout any one experiment. smooth suspension which upon microscopic inspection The titration of the Coombs serum, with minimal mod- yielded definite but scattered clumps was designated +. ification, was conducted in accordance with the suggestion Rare microscopic clumps in a grossly negative prepara- of Evans and Duane (11). Serial dilutions of the tion were ignored and the test was called negative. Since canine serum anti-serum with saline were prepared. To the + designation is questionable, no significance is given 0.1 cc. of the diluted serum was added 0.05 cc. of a 2 to it in this presentation. The microscopic appearances per cent suspension of washed erythrocytes. The mix- are depicted in Figure 1. ture stood at room temperature for 30 minutes, then was FIG. 1. MICROSCOPIC APPEARANCE OF THE VARIOUS GRADES OF AGGLUTINATION INDUCED BY THE DI- RECT COOMBS TEST FOLLOWING PHENYLHYDRAZINE. (A) DEPICTS THE MICROSCOPIC APPEARANCE OF A GROSS ± REACTION; (B) A 1 + REACTION; (C) A 2 + REACTION; AND (D) A 3 + REACTION 1702 E. E. MUIRHEAD, M. GROVES, AND S. BRYAN centrifuged at 500 to 1000 rpm. for one minute and evalu- above. The hemolyzed erythrocytes were prepared as ated for clumping. follows: the erythrocytes from the normal animals used The trypsin-treated erythrocytes were prepared by a were washed three times with abundant saline, a final sus- modification (32) of the description of Morton and Pickle pension of erythrocytes in saline was prepared with the (33). A 2 per cent solution of trypsin (Difco 1: 250) in same concentration of erythrocytes as originally present buffered saline (pH 7.3) was stcred at - 200 C. in small in the blood, this suspension was hemolyzed by repeated aliquots until used. A 1: 10 dilution of this 2 per freezing and thawing. Altogether there were eight tests cent solution in buffered saline was used as a working against the sera and eight tests against the hemolyzed solution. To 4.5 ml. of a 10 per cent suspension of fractions. In all instances the indirect Coombs test was washed RBC was added 0.5 ml. of the diluted trypsin negative. solution. This mixture was incubated for 10 minutes at The influence of hemolysis per se on the Coombs test 370 C., centrifuged at 2500 rpm. for 10 minutes and the was appraised also by the injection of 300 cc. of hemo- supernatant decanted. The cells were re-suspended to 2 lyzed blood, obtained by repeated freezing and thawing, per cent suspension in the buffered saline before using. into a previously unused normal dog. The effect of this The test for "incomplete antibodies" using a colloid procedure on the direct Coombs test, Heinz body con- medium (34) was performed by the use of a final sus- centration and mechanical fragility of the recipient's pension of erythrocytes in 22 per cent human albumin circulating erythrocytes was noted periodically for one solution according to the following steps: The 22 per week. These tests yielded negative results. cent solution was made up from a 25 per cent solution These observations indicated that hemolysis per se of human albumin in buffered diluent (E. R. Squibb and did not cause a false positive Coombs test with the ca- Sons, New York) by adding buffered saline (pH 7.3 to nine antiserum prepared in this laboratory. 7.5). To 0.1 cc. of 22 per cent albumin in a 13 X 75 mm. test tube was added 1 drop of a 2 per cent suspension of RESULTS the test cells which had been washed three times in abun- Direct Coombs test dant saline. This was incubated at 37° C. for 30 min- utes, centrifuged at 500 to 1000 rpm. for 1 minute and ex- a) Results znith the undiluted Coombs serum amined macroscopically for clumping.
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